Nasal transmission of equine parvovirus hepatitis

Abstract Background Equine parvovirus hepatitis (EqPV‐H) is highly prevalent and causes subclinical to fatal hepatitis, which can occur in outbreaks. Whereas iatrogenic transmission is well documented, the mode of horizontal transmission is not known. The virus is shed in nasal, oral and fecal secretions, and PO transmission has been reported in a single horse. Hypothesis/Objective Investigate the efficiency of PO and nasal transmission of EqPV‐H in a larger cohort. Methods Prospective experimental transmission study. Eleven EqPV‐H‐negative horses were inoculated with 5 × 106 genome equivalents EqPV‐H. Serum PCR and serology for EqPV‐H were performed weekly and monthly, respectively. Horses first were inoculated PO, and then intranasally 8 weeks later. Results No horse became viremic or seroconverted within 8 weeks after PO inoculation. After intranasal inoculation, 5 horses became viremic within 6 to 12 weeks and seroconverted within 10 to 19 weeks. After a period without monitoring from 12 to 19 weeks postinoculation, another 5 horses were found to be viremic at 19 to 22 weeks. The second set of 5 horses could have been infected by horizontal transmission from the first 5 because of cohousing. Conclusions and Clinical Importance We demonstrated that EqPV‐H can be transmitted nasally. The prolonged eclipse phase before detectable viremia indicates biosecurity measures to control spread could be impractical.


| Inoculations and sampling
Unfasted horses were inoculated PO with 5 Â 10 6 genome equivalents (GE) EqPV-H in equine serum, diluted in phosphate-buffered saline (PBS) to a final volume of 5 mL. Eight weeks later, horses were inoculated intranasally (IN) with the same inoculum using a 6-in. soft rubber catheter with diffuser tip. Horses that had not become viremic or seroconverted were challenged IV to document susceptibility to EqPV-H infection. Serum EqPV-H qPCR was performed weekly, and LIPS serology approximately monthly.

| Quantitative polymerase chain reaction
Viral nucleic acids were extracted from serum with Qiagen Viral RNA Mini kit (catalog no. 52906) according to the manufacturer's instructions. No DNase treatment was applied. The PCR was performed using primers EqPV-H q VP1 F15/R15 as previously described. 1 All PCR reactions were run on the QuantStudio 3 and analyzed on the QuantStudio 3 software (ThermoFisher). Suspect positive samples near the limit of detection were confirmed by submission to the New York State Animal Health Diagnostic Center (AHDC), which uses the PCR method described previously. 2
Blood smears were examined manually to confirm automated results.

| Serology
Antiviral protein 1 (VP1) immunoglobulin G antibodies were detected in serum by LIPS assay, as previously described, 16 with the following modifications. Briefly, the pREN2-EqPV-H plasmid (a kind gift from Dr. Peter Burbelo, National Institutes of Health, Washington DC) was transformed into TOP10 Escherichia coli, which were transfected into Cos1 cells. The renilla luciferase-VP1 antigen (Ruc-Ag) was harvested as a cell lysate using a renilla luciferase assay system lysis buffer (Promega). Serum samples, tested in duplicate, were diluted in buffer A (50 mM Tris, pH 7.5, 100 mM NaCl, 5 mM MgCl 2 , 1% Triton X-100), incubated with Ruc-Ag for 1 h, followed by incubation with pro-

| Statistics
Descriptive statistics were performed.

| Overview experimental inoculation study
We performed an experimental inoculation study using 11 horses that were 9 mares and 2 geldings between the ages of 3 and 14 years old and of various breeds (9 Thoroughbred, 1 Quarter Horse, and 1 Appendix Quarter Horse). All horses were healthy by physical examination, CBC, and serum biochemistry, and confirmed to be EqPV-H negative by serum qPCR and LIPS serology at the start of their 8-week quarantine and again at the start of the inoculation study.
F I G U R E 1 Study timeline. Horses were enrolled in 2 groups: group A, 9 horses; group B, 2 horses. Before inoculations, horses were screened by physical examination, serum biochemistry, CBC, serum qPCR, and LIPS serology. Horses were quarantined for 8 weeks, and then were inoculated with 5 Â 10 6 genome equivalents (GE) EqPV-H in horse serum PO. They were inoculated IN 8 weeks later, and monitored an additional 8 weeks. (A) In group A, horses were given routine veterinary care 8 weeks after IN inoculation, followed by a 4-week rest period. Then, 12 weeks after IN inoculation, the herd developed an upper respiratory tract infection and monitoring was suspended for 7 weeks. Weekly monitoring was restarted at week 19, after horses clinically recovered. Susceptibility to EqPV-H infection was later confirmed by IV inoculation of the single horse that had not been infected. (B) In group B, both horses became infected within 10 weeks after IN inoculation. Gray horse, EqPV-H qPCR negative; black horse, EqPV-H qPCR positive Nine horses (group A) were enrolled and housed together, and an additional 2 horses (group B; horses MA and SIG) were enrolled later and housed together ( Figure 1).

| Group A inoculations and results
Group A (n = 9) horses were inoculated first PO with 5 Â 10 6 GE EqPV-H in equine serum, diluted in PBS to a final volume of 5 mL.
Serum EqPV-H qPCR was performed weekly for 8 weeks and LIPS serology at 0 and 8 weeks. None of the horses became viremic and all were seronegative 8 weeks after PO inoculation (February 4th; Figure 1A). All 9 horses then were inoculated IN with the same inoculum as described above, using a 6-in. soft rubber catheter with diffuser tip, and their heads were held elevated for approximately 30 seconds after inoculation. Again, serum EqPV-H qPCR was performed weekly for 8 weeks and LIPS serology approximately monthly. None of the 9 horses from group A became viremic by the end of the 8-week monitoring period after IN inoculation (April 5th; Figure 1A). Intravenous inoculation was planned as the next step to demonstrate the horses were susceptible to EqPV-H. The study was suspended until 1 month after resolution of clinical signs to allow the immune response to return to baseline.

| Group B inoculations and results
As with group A, group B (n = 2) horses were inoculated first PO and then IN, and monitored as described above. The horses neither became viremic nor seroconverted at 8 weeks after PO inoculation on April 5th ( Figure 1B). However, they did develop viremia 6 and 10 weeks after IN inoculation ( Figure 1B), which fits within the time

| DISCUSSION
Our study expands understanding of the transmission modes of EqPV-H. Iatrogenic transmission of this virus by administration of equine origin blood products has been well documented. 16 Additional methods of natural horizontal or vertical transmission must occur as well, because EqPV-H is transmitted among horses that have not received such treatments. 3 Oral, nasal, and fecal shedding during acute EqPV-H infection has been demonstrated, 1 indicating inhalation or ingestion as plausible transmission routes. Here, we observed nasal transmission and found no evidence of PO transmission, which contradicts our previous study where we reported PO but not intranasal transmission in 1 of 2 horses. 1 In that study, we first inoculated both horses IN, and after a follow-up period of 8 weeks during which both horses did not become viremic, we proceeded with PO inoculation.
Based on our present finding of a prolonged eclipse phase before detectable viremia after nasal transmission, we propose that the 1 horse that became infected 4 weeks after PO inoculation actually was infected from the IN inoculation that occurred 12 weeks earlier. The finding that transmission can occur by nasal exposure has important implications for biosecurity and vaccine design. Ideally, isolation of EqPV-H-infected individuals could be utilized to prevent transmission on a premise. However, doing so presents multiple challenges.
First, EqPV-H shedding has been documented for at least 10 weeks after acute infection. 1 Second, horses can be chronically infected for years, 16,24 and shedding during chronic infection has not yet been evaluated. Third, our study demonstrated a prolonged incubation period of up to 12 weeks before detectable viremia. Thus, both isolation of infected horses and monitoring of in-contact horses would require separation and repeated sampling, respectively, for many weeks or months. Vaccination provides an attractive alternative when an effective biosecurity approach to limit viral spread is impractical. Because many effective vaccines are available for various parvoviruses, it appears feasible also to develop an effective vaccine for EqPV-H. Based on our findings of transmission via nasal mucosal surfaces, we propose that a vaccine inducing robust local immunoglobulin A responses in the nose could be highly effective. 35

CONFLICT OF INTEREST DECLARATION
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